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Erin Bedford
To cite this version:
Erin Bedford. Gold surface nanostructuring for separation and sensing of biomolecules. Chemi-cal Physics [physics.chem-ph]. Université Pierre et Marie Curie - Paris VI; University of Waterloo (Canada), 2016. English. �NNT : 2016PA066527�. �tel-01529577�
Université Pierre et Marie Curie
University of Waterloo
ED 397
Laboratoire de Réactivité de Surface / Equipe de recherche
Gold surface nanostructuring for
separation and sensing of biomolecules
Par Erin Bedford
Thèse de doctorat de Physique et Chimie de Matériaux (UPMC) et
de Chemical Engineering (Nanotechnology) (UW)
Dirigée par Mme Claire-Marie Pradier et M. Frank Gu
Présentée et soutenue publiquement le 15 novembre 2016
Devant un jury composé de :
M. Rabah Boukkheroub, Directeur de recherche, IEMN
Mme Marie-Hélène Delville, Directrice de recherche, ICMCB
M. William Anderson, Professeur, University of Waterloo
Mme Christine Ménager, Professeur, UPMC
Mme Souhir Boujday, Maître de Conférences, UPMC
M. Frank Gu, Associate professor, University of Waterloo
Mme Claire-Marie Pradier, Directrice de recherche, UPMC
Rapporteur
Rapporteuse
Examinateur
Examinatrice
Directrice de thèse
Directeur de thèse
Directrice de thèse
Author’s Declaration
This research proposal consists of material all of which I authored or co-authored: see Statement of Contributions included in the thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners.
Statement of Contributions
Chapter 2 is taken from a published research article: Bedford, E. E., Boujday, S., Pradier, C. M. & Gu, F. X. Nanostructured and spiky gold in biomolecule detection: improving binding efficiencies and enhancing optical signals. RSC Adv. 5, 16461–16475 (2015). The chapter is my own work with valuable discussions and edits provided by Frank Gu, Claire-Marie Pradier, and Souhir Boujday.
Chapter 3 is taken from a published research article: Bedford, E. et al. An Experimental and Theoretical Approach to Investigate the Effect of Chain Length on Aminothiol Adsorption and Assembly on Gold. Chemistry - A European Journal 21, 14555–14561 (2015). Frederik Tielens performed the DFT modeling and analysis. Christophe Méthivier performed the XPS analysis. I performed the experimental work, data analysis and wrote the article in collaboration with Souhir Boujday and with input from the other authors.
Chapter 4 is taken from a published research article: Bedford, E. E., Boujday, S., Humblot, V., Gu, F. X. & Pradier, C.-M. Effect of SAM chain length and binding functions on protein adsorption: β-lactoglobulin and apo-transferrin on gold. Colloids
and Surfaces B: Biointerfaces 116, 489–496 (2014). I performed the experimental work
except for the XPS analysis, which was done by Vincent Humblot, and wrote the article, in collaboration with Souhir Boujday. Claire-Marie Pradier and Frank Gu provided valuable input and discussions regarding the experiments and the manuscript.
Chapter 5 includes experimental work performed by Elaine Huang and by myself. I performed all experimental planning, analyses, and writing, with valuable discussions and edits provided by Frank Gu, Claire-Marie Pradier, and Souhir Boujday.
Chapter 6 is entirely my own work, with valuable discussions and edits provided by Frank Gu, Claire-Marie Pradier, and Souhir Boujday.
Abstract
Detecting biomolecules for disease diagnosis, food and environmental safety, and biological research is challenging. After time-intensive purification steps, the biomolecule still has to be detected, often at extremely low concentrations. There is a push in the field of biomolecular detection for methods that are faster and easier to use than current methods, while still maintaining the high standard of sensitivity and selectivity required for accurate testing. Nanomaterials are on the same size scale as the biomolecules being detected, thus are interesting tools for advanced detection methods. In this work, we focus on biomolecular detection methods that use gold surfaces, as gold is biocompatible, easily functionalized, and can exhibit interesting optical properties that can be harnessed for sensitive detection. The goal is to work towards improved methods of gold surface-based biomolecular detection by studying and using different nanotools: self-assembled monolayers (SAMs) of alkanethiols and nanostructured gold shells on magnetic particles.
Gold surfaces are commonly functionalized with SAMs of alkanethiols for applications in biosensing. Highly selective biosensing often involves immobilizing biomolecules on a surface, but obtaining high detection efficiencies requires controlling their orientation, dispersion, and density. Functionalizing sensing surfaces with SAMs can offer control over biomolecule binding without dramatically affecting the proximity of the biomolecules to the surface. Despite how often SAMs are used, we still lack a complete understanding of the process of formation and how different SAMs influence biomolecule binding and recognition. We studied SAMs made from short-chain (cysteamine, CEA) and long-chain (11-mercaptoundecylamine, MUAM) amine-terminated alkanethiols using surface IR spectroscopy, x-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) modeling. As expected, the longer chains formed a more ordered SAM than the shorter chains, but in addition, XPS showed that the sulfur binding environments differed for chains of different lengths. DFT modeling
further showed that surface reconstruction upon binding occurred differently for long compared with short chains. For the short chain alkanethiols, the thiol–gold interface governs the layer, as CEA binds more strongly with the mechanism being closer to single-molecule adsorption than self-assembly, whereas for long chains, the thiol-gold interface is less influential as interactions between alkyl chains drive the system to self-assembly, leading to a more ordered layer.
To study the dependence of protein binding and subsequent recognition on alkanethiol self-assembled monolayers (SAMs), we investigated adsorption of two proteins on amine-terminated SAMs with different chain lengths and different binding methods using surface IR spectroscopy and atomic force microscopy (AFM). We found that protein immobilization varies with SAM chain length and is also influenced by the presence of a cross-linker. The presence of a rigid cross-linker favours the binding of proteins on long chain SAMs, while the effect is almost nonexistent on shorter chains. In addition, the presence of the cross-linker induces a better dispersion of the proteins on the surfaces, regardless of the length of the thiols forming the SAMs.
Nanostructured gold has interesting optical properties for use in biomolecular detection. In the second part of this work, we synthesized spiky gold shells on magnetic particles for combined magnetic separation and surface enhanced Raman spectroscopy (SERS) detection of biomolecules. Magnetic particles are often used to separate particular biomolecules from a physiological sample. By coating the particles with a gold shell that can act as a SERS substrate, our goal is to enable the subsequent detection of the separated biomolecules without requiring additional binding steps.
The particle cores are made from controlled aggregates of superparamagnetic nanoparticles; the resulting particles are large enough to be quickly separated from solution, but still show superparamagnetic behavior and therefore can be easily redispersed in solution for subsequent biomolecule binding and washing steps. The cores are coated with silica to protect the magnetic cores and to facilitate subsequent
synthesis of the gold shell. The gold shell is made by binding small gold nanoparticle seeds to the silica surface, then using a chemical reduction method with cetyltrimethylammonium bromide (CTAB) as a stabilizer and structure-directing agent to grow the seeds into larger anisotropic gold/silver particles that combine into a shell. We explored different silica functionalization methods (bare silica, amine groups, short-chain thiol groups and long-short-chain thiol groups) to determine which method resulted in the best conditions for gold seed binding and growth into gold nanostructures. The gold shells can act as SERS substrates, enhancing the signal of a model Raman probe molecule, 2-mercaptopyrimidine, by a factor on the order of 104.Varying the growth
bath conditions (concentration of CTAB and time in growth bath) changes the morphology of the shells, as well as the degree of SERS enhancement and the stability of the particles.
As a proof-of-concept, we used the particles to detect oligonucleotide hybridization. We bound thiolated oligonucleotide probes to the gold coatings and measured the SERS signal. We were able to measure relative concentrations of oligonucleotides bound and showed that a single particle provides sufficient enhancement to detect bound oligonucleotides. We also studied the decrease in signal that occurs as the distance from the surface increases and found that at an estimated distance of ~7-10 nm from the surface, DNA bases were no longer detectable. An initial experiment comparing pre-hybridized oligonucleotides bound to the surface showed that directly detecting differences between single-stranded and double-stranded DNA would be challenging, so we used a probe designed to harness the distance dependence of the signal instead. Before hybridization, the probe is expected to be in a hairpin conformation with the Raman tag near the surface. After hybridization, the probe straightens, drawing the tag away from the surface and leading to a decreased signal intensity. We successfully demonstrated that the particles could be used to detect DNA hybridization without the use of an extrinsic label.
Résumé
La détection de molécules biologiques à des fins de diagnostic médical, de sécurité alimentaire, environnementale et de recherche biologiquerelève d’une grande difficulté. En effet, après des étapes de purification chronophages, la molécule biologique doit encore pouvoir être détectée, et ce à des concentration souvent extrêmement faibles. Par conséquent, la recherche pour le développement de méthodes de détection rapide, faciles à mettre en œuvre, avec une grande sélectivité et sensibilité fait l’objet d’un intérêt grandissant. Les nanomatériaux étant de la même échelle que les biomolécules à détecter sont donc des candidats idéaux à inclure dans des techniques de détection avancée.
Dans ces travaux de thèse, nous nous sommes concentrés sur des méthodes de détections biomoléculaires mettant en œuvre des surfaces en or car l’or présente les avantages d’être biocompatible, facile à fonctionnaliser et démontre des propriétés optiques pouvant être sollicitées pour de la détection sensible. L’objectif est de développer des méthodes de détection biomoléculaire basées sur des surfaces en or à travers l’étude et l’utilisation de différents outils nanométriques : des monocouches auto assemblées (self assembled monolayers (SAM) en anglais) d’alcane-thiolet des coquilles d’or nanostructurées sur des particules magnétiques.
Pour des applications en biodétection, il est commun d’utiliser des surfaces d’or fonctionnalisées avec des SAMs d’alcane-thiol. Afin que la biodétection soit hautement sélective, les biomolécules doivent généralement être immobilisées sur une surface. De plus, l’orientation, la dispersion, et la densité de ces biomolécules doivent être contrôlées pour une haute efficacité de détection. La fonctionnalisation de surfaces de détection avec des SAMs permet de maîtriser l’immobilisation des biomolécules sans modifier de manières significatives la proximité des biomolécules à la surface. Cependant, malgré de nombreuses études sur les SAMs, des interrogations subsistent concernant le mécanisme
de formation de la monocouche ainsi que l’influence du type de SAM sur l’immobilisation et sur la reconnaissance.
Nous avons étudié des SAMs à base de chaînes courtes (cysteamine, CEA) et de chaînes longues (11-mercaptoundecylamine, MUAM) des alcane-thiols avec une fonction amine terminale. Les méthodes de caractérisation utilisées dans ces études incluent la spectroscopie infrarouge de surface, la spectroscopie de photoelectrons X (X-ray photoelectron spectroscopy, XPS, en anglais) ainsi que la chimie théorique (théorie de la fonctionnelle de la densité ou density functional theory, DFT, en anglais). Comme attendu, les chaînes longues forment des SAMs plus ordonnées que celles obtenues par des chaînes courtes. De plus, l’XPS montre que l’environnement des soufres immobilisés diffère en fonction de la longueur des chaînes alcanes. La modélisation DFT montre, de plus, que la longueur de chaîne influence la reconstruction de la surface. Pour les chaînes courtes d’alcane-thiol, l’interface thiol/or influence la formation de la couche. En effet, la cysteamine s’accroche plus fortement à travers un mécanisme plus proche de l’adsorption moléculaireque d’un mécanisme d’auto-assemblage. D’autre part, pour les chaînes longues, l’interface thiol/or est moins prédominante dans la formation de la monocouche car les interactions entre les chaînes alcanes poussent le système à s’auto-assembler ce qui conduit à la formation d’une couche plus ordonnée.
Afin d’étudier l’effet des SAMs d’alcane-thiol sur l’accroche de protéines (et donc la reconnaissance qui en résulte), nous avons mené une étude sur l’adsorption de deux protéines sur des SAMs comprenant un groupe amine terminal. Les caractérisations menées comprennent la spectroscopie IR de surface ainsi que la microscopie à force atomique (atomic force microscopy, AFM, en anglais). Les résultats montrent que l’immobilisation des protéines varie en fonction de la longueur de la chaîne des SAMs et en fonction de la présence d’un réticulant. La présence d’un réticulant rigide favorise l’accroche de protéines sur des SAMs avec une chaîne longue alcane alors que l’effet est quasi-inexistant pour des SAMs de chaînes courtes. De plus, la présence d’un réticulant
implique une meilleure dispersion des protéines à la surface, indépendamment de la longueur de chaînes.
L’or nanostructuré possède des propriétés optiques intéressantes pour des applications en détection biomoléculaire. Dans la seconde partie des travaux, nous avons synthétisé des coquilles d’or nanostructurées sur des particules magnétiques afin de combiner la séparation magnétique et la détection de biomolécules par la spectrométrie Raman exaltée de surface (surface enhanced Raman spectroscopy, SERS, en anglais). Les particules magnétiques sont souvent utilisées afin de séparer les biomolécules particulaires de l’échantillon physiologique. Notre objectif est ainsi de permettre la détection, sans étape d’accroche subséquent,de biomolécules séparées, en déposant une coquille d’or pouvant agir en tant que substrat SERS sur les particules.
Le coeur des particules est constitué d’agrégats contrôlés de nanoparticules superparamagnétiques; les particules résultantes sont assez larges pour être séparées rapidement de la solution, mais conservent un comportement paramagnétique et peuvent donc être facilement re-dispersées en solution pour des étapes d’immobilisation de biomolécules et de lavages. Les noyaux sont recouverts de silice afin de protéger les coeurs magnétiques et de faciliter la synthèse subséquente de la coquille d’or.
Afin de former la coquille d’or, des initiateurs d’or sont déposés sur la surface de silice. Cette étape est suivie d’une réaction de réduction chimique avec le cetyltrimethylammonium bromide (CTAB) qui joue le rôle de stabilisateur et d’agent structurant permettant de contrôler la croissance des initiateurs en particules d’or et d’argent anisotropiques qui peuvent s’assembler en coquille.
Nous avons exploré plusieurs méthodes de fonctionnalisation de silice (silice pure, groupe amine, groupe de chaîne courte de thiol et groupe de chaîne longue de thiol) afin de déterminer quelle méthode serait la plus appropriée pour l’accrochage des initiateurs d’or ainsi que pour leur croissance en nanostructure d’or. Les coquilles d’or peuvent se comporter comme des substrats SERS permettant ainsi d’augmenter le signal d’une
sonde Raman, la 2-mercaptopyrimidine, par un facteur de l’ordre de 104. La
morphologie des coquilles, le degré d’augmentation du degré de SERS ainsi que la stabilité des particules se sont montrés influencés par les conditions de croissance (la concentration de CTAB et la durée).
Afin d’établir une preuve de concept, nous avons utilisé les particules nanostructurées pour la détection de l’hybridation d‘oligonucléotides. Des sondes d’oligonucléotides thiolés ont été déposées sur des dépôts d’or et le signal SERS résultant a été mesuré. Des concentrations relatives d’oligonucléotides ont pu être mesurées et il a été montré qu’une particule seule présente un signal suffisamment élevé pour une détection d’oligonucléotide. Nous avons aussi étudié l‘effet de l’augmentation de la distance à la surface sur la baisse de signal. Les résultats montrent qu’à une distance de 7 – 10 nm de la surface, les bases ADN n’étaient plus détectables. Une première expérience, comparant les oligonucléotides pré-hybridés accrochés à la surface, montra que la détection directe des différences entre l’ADN simple brin et double brin était compliquée. Nous avons donc utilisé une sonde spécifique (Cy5) afin de maîtriser la dépendance du signal à la distance. Avant hybridation, nous nous attendions à ce que la sonde soit dans une conformationen épingle à cheveuxquand la sonde Raman est près de la surface. Après hybridation, l’oligonucléotide se redresse, poussant la sonde loin de la surface et conduisant à une diminution de l’intensité du signal. Nous avons ainsi montré que ces particules pouvaient être utilisées pour la détection d’hybridation de l’ADN et, ce, sans l’utilisation d’une sonde extrinsèque.
Acknowledgements
I have had the enormous pleasure to have spent the last few years working in two excellent institutions: l’Université Pierre et Marie Curie (UPMC) and the University of Waterloo (UW). The opportunity to do a cosupervised/cotutelle PhD was provided by the IDS-FunMat program. I’m also grateful to my funding sources: to UPMC and UW, the Natural Science and Engineering Research Council of Canada (NSERC), the French Embassy in Canada/France-Canada Research Fund (FCRF), and the Waterloo Institute for Nanotechnology (WIN).
To my supervisors, Prof. Frank Gu, Dr. Claire-Marie Pradier, and Prof. Souhir Boujday—thank you. Thank you for helping to guide me throughout my work and for supporting me on the path to becoming an independent researcher. You guided me and provided me with your valuable expertise, but always left me with the freedom to choose my own path. This has made me not just a better researcher, but also a better person. Your encouragement in both the day-to-day work and in my future career choices has been amazing. You have my sincere gratitude.
Thank you as well to my committee members, Dr. Rabat Boukherroub, Dr. Marie-Hélène Delville, Prof. William Anderson, and Prof. Christine Ménager for agreeing to review my thesis and share their expertise.
Another thanks goes to Tornado Spectral Systems for lending us a Raman spectrometer that was used extensively in experiments and especially to Yusuf Bismilla for helping to set this up.
Many thanks to all members of the Laboratoire de Réactivité de Surface. Your help in both the lab and in navigating life in Paris was amazing—I couldn’t have done it without you. You put up with my not-so-great French and helped me to reach the point where (I’m pretty sure) we could understand each other without resorting to English. A
Vincent Humblot, Jean-Marc Krafft, Christophe Méthivier, Christophe Calers, and Sandra Casale for their help obtaining and analyzing data, and the indispensable discussions surrounding it. Special thanks also to Rickielle, Kim, Cédric, Jane, Maroua, Noémie, Antoine, Jessie, Colin, and Catarina for the support in the lab and the great times spent over coffee or a pint.
Many thanks as well to the Waterloo lab members. I’ve always considered myself ridiculously lucky to be able to turn around at my desk and ask “hey guys, do you have a second?”, throw out an idea, and get amazing feedback. And thanks for the fun times as well—if the origami buckyball goes missing from the office, the first place to check should probably be my living room. Special thanks to Sandy for putting up with me staring into “space”, i.e. the side of his head, to Jasper for the bike rides and jogs, to Sarah for the support and friendship (and for making it so that I wasn’t surrounded by only boys), to Elaine for being an amazing co-op student, to Paul, Perry, and Jackson for making and sending me particles, and to Mohit, Tim, Stuart, and Kuba for the always helpful chats and inspiration.
I’d also like to thank those in both universities who helped me to sort out the paperwork and day-to-day administrative tasks associated with doing a PhD, especially with doing a PhD in two countries. In the LRS, special thanks to Sabine Mendes, Sabine Même, Annie Mettendorf, and Sonia M’Barek. At UW, special thanks to Judy Caron and the others in the chemical engineering and grad studies offices.
While my above thank-you’s certainly include those who I consider not just co-workers but also friends, I’d also like to thank my friends outside of the lab who helped to maintain my sanity. To my Waterloo IDS-FunMat crew and friends—Edgar, Uyxing, Mylène, Dan, Cam, An, Jiang—thanks for the lunches, coffees, w(h)ine and cheeses, and general support and fun. Thanks as well to all my MEC friends—especially to Heather and Michèle for the semi-marathon training and delicious meals, to Sophie for the course notes and company, and to Guillaume for the support, encouragement, and all
around great times. And thanks to my friends in Canada—to Richard for always, always being there, to Helen for the listening ear and the belaying hand, and to the Grad House gang when the best option seems to be (in celebration or otherwise) a beer.
To my family—thank you for the never-ending support. Mom and dad, I guess you’ve done a pretty good job of raising me to feel confident supporting me in whatever decisions I’ve made—thank you, from the bottom of my heart. Don, your support has also been amazing. Taylor, my little sister, thanks for being wonderful.
Table of Contents
Author’s Declaration ... iii
Statement of Contributions ... iv
Abstract ... v
Résumé ... viii
Acknowledgements ... xii
List of Figures ... xviii
List of Tables ... xxiv
List of Abbreviations ... xxv Chapter 1 : Introduction ... 1 Overview ... 1 Outline ... 3 : Literature Review ... 5 Summary ... 5 Introduction ... 5 Benefits of Nanostructuring ... 8 Geometric Benefits ... 8
Enhancement of Optical Detection Methods... 12
Types of Gold Nanostructuring ... 18
Planar Surface Nanostructuring ... 19
Particle Nanostructuring ... 24
Conclusion ... 33
: An Experimental and Theoretical Approach to Investigate the Effect of Chain Length on Aminothiol Adsorption and Assembly on Gold ... 34
Introduction ... 35
Experimental Section ... 36
Materials ... 36
Methods ... 36
Techniques ... 37
Results and Discussion ... 39
PM-IRRAS characterization of SAMs on gold surface ... 39
XPS characterization of SAMs on gold surface ... 40
DFT geometry optimizations and binding energies of the SAM systems .... 43
General discussion ... 47
Conclusions ... 48
: Effect of SAM chain length and binding functions on protein adsorption: β-lactoglobulin and apo-transferrin on gold ... 50
Summary ... 50 Introduction ... 51 Experimental Section ... 53 Materials ... 53 Methods ... 54 Characterization techniques ... 56 Results ... 57
SAM formation and cross-linker binding ... 57
Protein immobilization ... 59
Discussion ... 64
Conclusion ... 66
: Synthesis and characterization of nanostructured gold coatings on magnetic particles ... 67
Summary ... 67
Experimental Section ... 70
Materials ... 70
Methods ... 71
Characterization techniques ... 75
Results and Discussion ... 77
Magnetic properties ... 77
Gold shell growth: Influence of silica-iron oxide core functionalization ... 81
Gold shell growth: Influence of growth bath conditions ... 93
Particle stability over time ... 96
Conclusion ... 99
: Nanostructured gold shells on magnetic particles for oligonucleotide detection ... 100 Summary ... 100 Introduction ... 100 Experimental Section ... 103 Materials ... 103 Methods ... 103
Results and Discussion ... 107
Oligonucleotide binding ... 107
Micro-Raman study of individual particles and small clusters ... 111
Raman study of oligonucleotide “rulers” ... 116
DNA hybridization detection ... 120
Conclusion ... 127
: Conclusions and Perspectives ... 128
References ... 131
Appendix A : Apo-transferrin data ... 146
List of Figures
Figure 2.1: General scheme of biosensors. Targets in a biological sample bind to receptors bound to a substrate. Signal transduction indicates target binding ... 6 Figure 2.2: Proposed model of the effect of nanostructuring on DNA binding and
hybridization. Nanostructured microelectrodes (NMEs) were used for electrochemical detection of DNA hybridization with and without nanotexturing. Reprinted with permission from ref49. Copyright 2010 American Chemical Society. 49 ... 10
Figure 2.3: Proposed model of the effect of nanostructuring on protein-antibody (biotin-IgG) interactions. Feature sizes similar to the size of the binding domains of IgG result in higher recognition (B, C). Feature sizes that are too small prevent recognition (A) and those that are too large result in random orientations (D). Reprinted with permission from ref 10. Copyright 2008 American Chemical Society.
... 12 Figure 2.4: Simulations of electromagnetic enhancement at a) nanogaps72, b) sharp tips73,
and c) combined sharp tips and nanogaps (bowtie nanoantenna)74. ... 14
Figure 2.5: General scheme of nanosphere lithography... 23 Figure 2.6: SEM images of gold nanostructured surfaces formed by electrodeposition for
different times: (A) 20 s, (B) 100 s, (C) 300 s, and (D) 600 s.54 ... 24
Figure 2.7: Examples of anisotropic particles from literature. Labels correspond to those in Table 2.1. ... 31 Figure 3.1: Cysteamine (CEA) and 11-mercaptoundecylamine (MUAM) SAMs formed
on gold surface (schematic representation) ... 37 Figure 3.2: PM-IRRAS spectra of: a) Au-CEA and b) Au-MUAM ... 40
Figure 3.3: High-resolution XPS S2p region for Au-MUAM (left) and Au-CEA (right) .. 42 Figure 3.4: Side views of the optimized geometry of a) CEA and b) MUAM SAMs ... 44 Figure 3.5: Top view of a) CEA SAM and b) MUAM SAM showing only the sulfur and
gold atoms. The sulfur atom (red) on the Au surface (yellow: bulk atoms, green: surface Au atoms, magenta: surface extracted Au atoms) ... 45 Figure 3.6: Top view of a) CEA SAM and b) MUAM SAM showing the relative
displacement (arrows) of the sulfur atoms (red). The gold atoms (green) are at a distance smaller than 2.69 Å from the Sulfur atoms ... 46 Figure 4.1: Schematic drawing of protein immobilization methods on SAMs made from
amine-terminated short-chain thiols (cysteamine, CEA) and long-chain thiols (11-mercaptoundecylamine, MUAM). (Top) Immobilization on surface amine groups following EDC/NHS activation of acid groups on proteins. (Bottom) Cross-linker binding and protein immobilization by reaction with amine groups on proteins. .. 55 Figure 4.2: PM-IRRAS spectra of Au-CEA, Au-CEA after PDITC binding, Au-MUAM
and Au-MUAM after PDITC binding. ... 58 Figure 4.3: PM-IRRAS spectra after β-lactoglobulin (a) and antibody recognition (b) on
CEA, MUAM, CEA-PDITC and MUAM-PDITC. ... 60 Figure 4.4: AFM images of gold-coated substrates following SAM formation and β-lactoglobulin binding. Upper images: Scan area: 10 µm x 10 µm, height scale: 40 nm. Lower images: Scan area: 1 µm x 1 µm, height scale: 10 nm. ... 62 Figure 5.1: Schematic diagram of spiky particle synthesis steps. Gold seeds are bound to
a silica-coated magnetite particle, then grown into gold spikes using a gold salt and CTAB bath solution. ... 71 Figure 5.2: Silica coatings were functionalized using different groups. Silica shells were
using MPTMS (SC-SH), and d) Long-chain thiol-coated, using MUA grafted onto amine-coated particles (LC-SH). ... 73 Figure 5.3: Magnetization curves of silica-coated iron oxide particles before (solid) and
after (dashed) gold/silver shell coating. The inset shows the small amount of hysteresis occurring at low magnetic fields ... 78 Figure 5.4: Measured opacity over time for water dispersions of particles at 1 mg
Fe3O4/SiO2 particles/ml (greater mass upon gold coating) in a 45 T/m gradient,
before (solid) and after (dashed) gold shell coating. ... 79 Figure 5.5: Interaction potential at short distances between two silica-coated iron oxide
particles (iron oxide core diameter = 224 nm, silica shell diameter = 336 nm) with a charge density of −0.9 e/nm2 in 10 mM concentration of 1:1 electrolyte (surface
potential Φ0 ≈ −73 mV). (Blue) no applied magnetic field—zero magnetization, (red)
remanent magnetization following removal of magnetic field, (green) applied magnetic field—saturation magnetization of 32 emu/g. ... 80 Figure 5.6: TEM images of particles after gold seed binding on a) bare silica, b) amine-functionalized silica, c) short-chain thiol-amine-functionalized silica, and d) long-chain thiol-functionalized silica ... 83 Figure 5.7: UV-Vis spectra of particles before and after gold seed binding ... 83 Figure 5.8: TEM images of spiky particles, with spiky nanostructures grown on a) bare
silica, b) amine-functionalized silica, c) short-chain thiol-functionalized silica, and d) long-chain thiol-functionalized silica. Insets show high magnification of spiky shells ... 85 Figure 5.9: UV-Vis spectra of spiky particles ... 86 Figure 5.10: Raman spectra of 2-mercaptopyrimidine with and without particles. Note
enhancement by particles (10 μM) compared with the unenhanced signal (10 mM). All samples used 50 μl of 2 mg/ml particles in 1 mM CTAB added to 3 ml of water. ... 89 Figure 5.11: Raman spectra of 2-mercaptopyrimidine (MPym) at different concentrations
with particles featuring spiky gold shells on bare silica-coated magnetite particles. All samples used 50 μl of particles in 1 mM CTAB added to 3 ml of water. Peaks with red labels correspond to peaks that increased after adding MPym and peaks with black labels correspond to peaks seen on particles before adding MPym— likely CTAB ... 90 Figure 5.12: Change in peak height of Raman signal corresponding to the Raman
reporter (MPym) at different concentrations. A fit to a Langmuir model (red line) shows saturation behavior (R2 = 0.98) ... 91
Figure 5.13: Change in peak heights corresponding to CTAB at different Raman reporter (MPym) concentrations. ... 92 Figure 5.14: a) TEM images showing representative particles at varying growth times
and CTAB concentrations and b) corresponding UV-Vis extinction spectra for particles synthesized at different growth times using (top) 10 mM and (bottom) 100 mM CTAB ... 94 Figure 5.15: a) TEM images and b) (i-iii) corresponding Raman spectra of particles in
167µM CTAB with i) spiky shells (EB-150-a), ii) bumpy shells (EB-150-b), and iii) nanoparticle spotted (EB-150-c), and iv) Raman spectra of 0.2M CTAB in water (1200x more concentrated than particle-enhanced measurements). ... 95 Figure 5.16: a) TEM images showing representative particles over time and b)
corresponding UV-Vis extinction spectra for (left) spiky particles and (right) bumpy particles over time ... 98
Figure 5.17: Raman spectra of (top) spiky and (bottom) bumpy particles in 167 µM CTAB ... 99 Figure 6.1: Oligonucleotides as received. TCEP is used to reduce them to thiol form. .. 107 Figure 6.2: SERS spectra of spiky gold-coated magnetic particles functionalized with a) i)
3-mercapto-1-propanol, and ii) oligonucleotides with sequence 5’-C12A3-3’-(CH2)3
-S-, b) i) 6-mercapto-1-hexanol-S-, and ii) oligonucleotides with sequence -S-(CH2)6
-5’-A3C12-3’ ... 109
Figure 6.3: SERS spectra of oligonucleotides (5’-C8A3C4-3’-S-) bound to particles using
different binding concentrations ... 110 Figure 6.4: Comparison of measured Raman intensity of peak at 1029 cm-1 and UV-Vis
intensity at 260 nm for oligonucleotides on particles using different binding concentrations ... 111 Figure 6.5: Micro-Raman (SERS) spectra of thiolated oligonucleotides bound to spiky
gold-coated magnetic particles on silicon. Each spectrum represents a separate measurement focusing on a) individual particles and b) small groups of particles (2-4) ... 113 Figure 6.6: a) TEM images of particles with different gold shell morphologies: i) super
spiky shells, ii) spiky shells, and iii) bumpy shells. b) Average micro-Raman (SERS) spectra of 7-10 measurements for (blue) individual particles, (solid red) small groups of particles, and (dashed red) small groups of particles normalized using the average number of particles in the measurements, which were i) 2.2, ii) 2.8, and iii) 2.4. c) Graphs highlighting average Raman spectra peak height similarity between signals from single particles and small groups of particles after adjusting for the average number of particles. No pairs of means show significant differences. Error bars indicate 95% confidence intervals. ... 115
Figure 6.7: Graphs highlighting average micro-Raman (SERS) spectra peak height differences between signals from particles of different morphologies. Significant differences are indicated by asterisks: (*) indicates p ≤ 0.05 and (***) indicates p ≤ 0.001. Error bars indicate 95% confidence intervals. ... 116 Figure 6.8: SERS spectra of oligonucleotide “rulers” bound to spiky gold-coated
particles. The rulers (i-vii) correspond to those listed in Table 6.2. Two spectra are shown overlaid for each oligonucleotide. ... 118 Figure 6.9: Normalized Raman peak intensity (I1338) for oligonucleotides with different
adenine group positions. The red line shows a fit to the data based on the distance dependence of the SERS signal. Error bars indicate a 95% confidence interval. .... 120 Figure 6.10: Scheme of hairpin probe with Raman tag, before and after hybridization to
target strand ... 121 Figure 6.11: a) SERS spectra and b) normalized SERS spectra (using intensity at 1087 cm -1) of i) oligonucleotide probes only, ii) oligonucleotide probes hybridized with
complementary oligonucleotides, and iii) oligonucleotide probes hybridized with non-complementary oligonucleotides ... 122 Figure 6.12: a) SERS spectra and b) normalized SERS spectra (using intensity at 1023 cm -1) of i) Cy5-tagged oligonucleotide probes only, ii) Cy5-tagged oligonucleotide
probes hybridized with complementary oligonucleotides, and iii) Cy5-tagged oligonucleotide probes hybridized with non-complementary oligonucleotides ... 123 Figure 6.13: a) Normalized SERS spectra and b) average peak height of i) Cy5-tagged
oligonucleotide hairpin probes, ii) hybridization with complementary oligonucleotides and iii) hybridization with non-complementary oligonucleotides. Error bars indicate 95% confidence intervals. ... 125
List of Tables
Table 2.1: Particles with nanostructured surfaces – method of synthesis and demonstrated application ... 32 Table 3.1: XPS-determined atomic percentages and S2p photopeak decompositions
corrected by Scoffield factors ... 41 Table 4.1: Change in area of amide I and II (1660 cm-1 + 1550 cm-1) peaks from PM-IRRAS
following binding and ratio of bound antibodies to corresponding proteins adjusted for mass (errors correspond to standard deviation from measurements of two to four separate chips). ... 60 Table 5.1: Separation times of particles, determined based on time required to reach 5%
and 1% of initial opacity (see dotted lines in Figure 5.4)... 79 Table 5.2: XPS results showing elemental breakdown of particle surfaces following
treatment ... 81 Table 5.3: XPS-determined atomic percentages following coating procedures in different
conditions ... 96 Table 6.1: Gold shell synthesis parameters and approximate concentration of
oligonucleotides bound ... 112 Table 6.2: Oligonucleotide sequences used as “rulers” ... 117
List of Abbreviations
APTES: (3-aminopropyl)triethoxysilane AFM: atomic force microscopy
AuNPs: gold nanoparticles CEA: cysteamine
CTAB: cetyltrimethylammonium bromide dsDNA : double-stranded deoxyribonucleic acid
EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDTA: ethylenediaminetetraacetic acid
NHS: N-hydroxysuccinimide MPym: 2-mercaptopyrimidine
MPTMS: (3-mercaptopropyl)trimethoxysilane MUA: 11-mercaptoundecanoic acid
MUAM: 11-mercaptoundecylamine NPs: nanoparticles
PDITC: p-phenylene diisothiocyanate
PM-IRRAS: polarization modulation infrared reflection absorption spectroscopy SAM : self-assembled monolayer
SEM: scanning electron microscopy
SERS: surface-enhanced Raman spectroscopy SPR: surface plasmon resonance
ssDNA : single-stranded deoxyribonucleic acid TCEP: tris(2-carboxyethyl)phosphine
TEM: transmission electron microscopy UV-Vis: ultraviolet-visible spectroscopy XPS: x-ray photoelectron spectroscopy
Chapter 1: Introduction
Overview
Biomolecular detection is used in a number of fields—to detect disease, to determine if a source of water is safe to drink, and to learn more about the form and function of organisms, for example. Biomarkers of disease or contamination can include proteins, nucleic acids, and other molecules specific to a given pathogen. Current methods of detection such as ELISA, polymerase chain reaction (PCR), and DNA/RNA microarrays work, but tend to require advanced labs with experienced personnel, as well as time and money. There is a push in the field of biomolecular detection/biosensing to develop methods that are fast and easy-to-use, while still exhibiting sufficient enough sensitivities and specificities for practical use.
Controlling small things means using small tools. As our ability to work with and control the microscopic biological world grows, our need for tools that can interact with this world does as well, which is where nanomaterials are playing an increasingly important role.1 In this work, we study several of these tools and use them to improve
biomolecular detection, specifically self-assembled monolayers (SAMs) of alkanethiols on gold and nanostructured gold surfaces. Gold surfaces are commonly used in biomolecular detection methods due to their biocompatibility, easy functionalization, and interesting electromagnetic properties that result at the nanoscale. SAM functionalization and nanostructuring of gold surfaces are important tools in the quest towards biomolecular detection that is more sensitive, faster, and easier to use.
Achieving high sensitivities in biosensing often involves using a surface-bound probe molecule to specifically detect the analyte (e.g. antibody, aptamer, complementary DNA). The nature of the probe binding—including factors such as probe density, conformation, and orientation—has a large influence on target binding efficiencies. Controlling the surface chemistry and other properties is therefore crucial to biosensor
function.2 Crowding effects due to high densities can prevent target molecules from
accessing binding sites on probe molecules.3-8 Good access to binding sites also requires
that the probes be bound in a suitable conformation and orientation.3,9,10 Self-assembled
monolayers of alkanethiols on gold are one tool that can be used to control probe molecule binding. In the case of proteins, for example, using surfaces functionalized with different groups changes which part of the protein is bound to the surface, thus which part of the protein is exposed and available for target binding.11 SAMs can also act
as diluting or spacing groups between probe molecules to control the density.3 While
SAMs on gold are commonly used in biosensing methods, optimization of these surfaces requires a better mechanistic understanding of SAM formation and biomolecule binding, which surface science methods can help provide.
Nanostructured gold is an increasingly common tool in biomolecular detection research because of its interesting optical properties.12-17 Surface-enhanced Raman spectroscopy
(SERS) uses nanostructured metallic surfaces (typically gold or silver) to enhance the electromagnetic signal of both the light incident to the surface and light scattered by molecules near the surface; the result is a signal intensity orders of magnitude greater than what is observed in ordinary Raman spectroscopy. SERS substrates are made in a variety of forms; commonly used substrates include planar surfaces with nanoscale metallic features, but SERS can also be done using gold or silver nanoparticles in solution. The greatest enhancements occur at hot-spots—sharp tips or nanogaps—so most current efforts involve synthesizing structures of this type that give a strong, reproducible enhancement.14,18
Multifunctional nanoparticles, where a single particle exhibits multiple functions (such as magnetism, fluorescence, catalytic, or optical properties), open up additional possibilities to improve biomolecular detection using nanomaterials. Here, we aim for combined separation and detection of biomolecules using magnetic cores with nanostructured gold shells that can act as SERS substrates. Biological samples are a complex mixture of biomolecules, so a first step in any attempt to work with a specific
biomolecule is to separate it from the rest. With the goal of a simple biosensing method with fewer steps, an ideal method would involve detecting the presence and quantity of the analyte directly after separation. Magnetic particles are commonly used for separating and concentrating specific biomolecules.19-23 Following magnetic separation,
the analyte is bound to the surface of the particles; directly detecting its presence without any further dissociation or binding steps is possible using the concept of multifunctional particles. We use magnetic particles coated with a nanostructured gold shell that can be used to see changes in the SERS signal upon binding of the target to the nanostructured gold surface.
Outline
We begin by reviewing methods of gold surface nanostructuring for biomolecular detection in Chapter 2. This includes both planar and particle-based methods and the benefits that have been shown in the literature.
The first part of the experimental work involves studying self-assembled monolayers of alkanethiols on planar gold surfaces and their influence on protein binding and recognition (Chapter 3 and Chapter 4). Self-assembled monolayers are often used in biosensing because they can easily form a layer of functional groups on the surface that can be used for biomolecule attachment. In the case of alkanethiols, the length of the carbon chain between the thiol group and the head group influences the properties of the self-assembled monolayer that forms. In Chapter 3, we investigate this effect in amine-terminated alkanethiols using x-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) modeling to compare long and short chain adsorption and assembly on gold surfaces. Long-chain alkanethiols are known to form more ordered layers than short-chain alkanethiols,24-27 but the nature of the gold-sulfur
interface is not yet completely understood. In this chapter, we propose a mechanism involving gold surface rearrangement that contributes to explaining the observed
differences in SAM organization with different chain lengths due to the nature of the gold-sulfur bond.
In Chapter 4, we study protein binding and recognition on different alkanethiol surfaces. We compared the binding of two proteins of different sizes (β-lactoglobulin and apo-transferrin) to SAMs of different chain lengths, with or without a cross-linker group, using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and atomic force microscopy (AFM). Protein binding amount and density is influenced by the nature of the SAM; consequently, recognition of the proteins by an antibody is also affected, due in part to the nanoscale arrangement of proteins on the surface.
In the second part of this work, we developed multifunctional particles for combined separation and detection of biomolecules (Chapter 5 and Chapter 6). The particles feature a nanostructured gold coating on a magnetic core; the gold coating acts as a surface-enhanced Raman spectroscopy (SERS) substrate, enhancing the Raman signal of molecules bound to their surface and the magnetic core allows the particles to be quickly separated from solution using magnetic forces. Chapter 5 describes their synthesis and the influence of varying conditions on their use as SERS substrates. In Chapter 6, we use the particles to detect DNA binding and hybridization, as a proof-of-concept. Using a Raman tagged oligonucleotide hairpin probe we can detect oligonucleotide hybridization by harnessing the distance dependence of the SERS signal.
Chapter 7 summarizes the results and discusses them from the perspective of the broader context of the field. We look at the implications and potential future directions of the work.
This PhD work was carried out in a collaboration between two universities: Université Pierre et Marie Curie (UPMC) in the Laboratoire de Réactivité de Surface (LRS) and the University of Waterloo (UW). The joint PhD collaboration was organized as part of the International Doctoral School in Functional Materials (IDS-FunMat), an Erasmus Mundus joint doctorate program.
: Literature Review
Summary
Nanostructured gold can improve the ability to detect biomolecules. Whether planar nanostructured surfaces or nanostructured particles are used, similar principles governing the enhancement apply. The two main benefits of nanostructured gold are improved geometry and enhancement of optical detection methods. Nanostructuring improves the geometry by making surface-bound receptors more accessible and by increasing the surface area. Optical detection methods are enhanced due to the plasmonic properties of nanoscale gold, leading to localized surface plasmon resonance sensing (LSPR), surface-enhanced Raman spectroscopy (SERS), enhancement of conventional surface plasmon resonance sensing (SPR), surface enhanced infrared absorption spectroscopy (SEIRAS) and metal-enhanced fluorescence (MEF). Anisotropic, particularly spiky, surfaces often feature a high density of nanostructures that show an especially large enhancement due to the presence of electromagnetic hot-spots and thus are of particular interest. In this review, we discuss these benefits and describe examples of nanostructured gold on planar surfaces and particles for applications in biomolecule detection.
Introduction
From medicine to environmental monitoring to food contamination protection, detection of biomolecules helps to protect our health and our environment. Over the past two decades, biosensor research has taken off, inspired by the success of the hand-held glucose sensors used by diabetic patients, but expanding into the detection of all types of biomolecules using a variety of transduction methods.28 No matter the method of
detection, high sensitivity is one of the constant goals within the field of biosensor research. Since many methods involve concentrating biomolecules on a surface for signal transduction, a key strategy to achieve high sensitivities is to optimize the
surfaces on which probes are bound so that a large number of analyte molecules can be bound and a sufficiently strong signal produced.
Affinity-based biosensors harness the specific affinity between certain biomolecules to detect the presence and quantity of a biomolecule. These make use of the same interactions that allow for currently used methods of detecting biomolecules such as immunoassays, which make use of the affinity between antigens and antibodies (ELISA, for example), and hybridization assays, which make use of the affinity between complementary nucleic acid strands (Southern and northern blot assays, for example). By using affinity interactions to specifically bind an analyte of interest—typically on a solid surface—they can be detected by various methods of signal transduction (Figure 2.1). Biomolecules are small, so harnessing their specific interactions requires tools of a comparable scale—a job that nanostructured gold fills well; gold is biocompatible, chemically stable, and can easily be functionalized.1,12,29,30
Figure 2.1: General scheme of biosensors. Targets in a biological sample bind to receptors bound to a substrate. Signal transduction indicates target binding
While electrochemical methods have traditionally been the most commercially successful biosensing methods, optical detection methods have also proved interesting
by offering highly sensitive, label-free detection— a primary example being the prevalence of surface plasmon resonance (SPR) detection in R&D.28 Optical methods of
detection make use of changes in the optical signal—absorption, luminescence, fluorescence, and plasmon resonance, for example—that occur upon binding. Again, nanostructured gold stands out as an interesting material, in this case, because of its interesting optical properties. Surface plasmons can be excited in metallic nanoparticles by specific wavelengths of light due to the confinement of electrons within the small particles.31,32 In gold nanoparticles, this surface plasmon resonance (SPR) frequency is in
the visible range, leading to the characteristic red colour of gold nanospheres and other colours in gold nanoparticles of different shapes and sizes. In addition to these distinct colours, the confined surface plasmons lead to enhanced electromagnetic fields at the particle surfaces. Anisotropic shapes, such as “spiky” tips, lead to particularly strong enhancements, often referred to as electromagnetic hot-spots.31,33-36 In localized surface
plasmon resonance (LSPR) sensing, biomolecule binding leads to a shift in gold nanoparticle absorbance, which is larger when biomolecules are bound to hot-spots compared with other areas of the gold nanoparticle surface.37-40 Other methods of optical
detection, such as surface-enhanced Raman spectroscopy (SERS),18,33,41 surface enhanced
infrared absorption spectroscopy (SEIRAS),33,42,43 and metal-enhanced fluorescence
(MEF)44 also show an enhanced signal due to this hot-spot phenomenon that can be
harnessed for biosensing applications.
Extraordinarily innovative methods have been used to form spiky gold nanostructures that exhibit the above features. Methods like electron beam lithography and atomic force microscopy can make precise structures that are extremely useful in studying the above phenomena, but the practical harnessing of these phenomena in biosensing will likely require simpler methods of nanostructure formation that can be done on a larger scale or that are more accessible to non-specialized laboratories.
followed by a (non-exhaustive) look at examples of methods used to nanostructure gold surfaces with a focus on chemical methods and those requiring less specialized equipment.
Benefits of Nanostructuring
Recent work has shown that there are numerous benefits to nanostructuring surfaces, including geometric benefits involving the position, orientation, and accessibility of immobilized biomolecules as well as enhancement of optical transduction methods. Our focus in this review will be on the geometric benefits and the enhancement of optical detection methods using nanostructured gold. This covers two different methods of enhancement: geometric optimization, by increasing the number of targets available for detection, and optical detection method enhancement, by increasing the sensitivity of the technique to a single recognition event.
Geometric Benefits Nucleic acids
DNA biosensors use the specific interaction between complementary strands of DNA bound to a surface and the DNA molecules of interest to detect the presence of a particular DNA sequence. Hybridization with DNA probes bound to a surface introduces new challenges compared with standard hybridization in solution. Hybridization efficiencies are reduced by electrostatic repulsion and steric hindrance between immobilized strands, and by non-specific adsorption of oligonucleotides to the surface.4-6,45
The idea that working with small biomolecules requires small tools has led to much research on the differences that occur between binding oligonucleotides to nanostructured surfaces and binding to planar ones. In particular, the surface curvature influences the interactions between bound probes, and consequently, the number of probes that can be immobilized on a surface. Researchers have found that the loading
density of thiolated DNA strands on sufficiently curved gold surfaces (for spherical particles, this means having a diameter less than 60 nm) can be an order of magnitude larger than on planar surfaces.46,47 This may be due to decreased electrostatic repulsion
due to increased deflection angles between strands on smaller particles.46,47 This theory is
supported by the fact that loading density also depends on salt concentration, where an increase in salt concentrations, up to a point, results in increased loadings due to its neutralizing effects on the negatively charged phosphate backbone of DNA.
While DNA loading is increased on curved surfaces, whether the highest possible DNA probe loading also results in optimal DNA hybridization is another question. While high DNA probe loadings are important in ensuring that a high number of targets are bound to a surface through hybridization, steric and electrostatic issues also become factors. On planar surfaces, optimal probe coverage for target binding involves a balance between a high number of probes for targets to be bound to and a low enough density that steric and electrostatic issues are not a problem. Several groups have shown that high probe densities reduce hybridization efficiencies.4-6 Irving et al demonstrated that the reduction
in hybridization efficiencies that results with high probe densities can be divided into regimes based on the main mechanism of hybridization suppression: an electrostatic suppression regime at lower salt concentrations and a packing suppression regime at higher salt concentrations.5
Do the same crowding issues occur on non-planar surfaces? We know that curved surfaces result in increased deflection angles between immobilized strands, so it would be expected that immobilization on convex surfaces at the same “footprint” densities as on planar surfaces would result in greater spacing between the accessible ends of strands, thus reduced electrostatic and steric barriers. This phenomenon has, in fact, been demonstrated experimentally. The Kelley lab has demonstrated that detection limits are decreased by several orders of magnitude when electrochemically detecting DNA hybridization on nanostructured palladium48-50 or gold51-53 compared with smooth
favorable geometries for hybridization (Figure 2.2) by showing that the greatest enhancements occur with fine nanostructuring (20-50 nm)—a similar length scale to the immobilized oligonucleotides (5-10 nm)50—and by showing that higher hybridization
efficiencies occurred on nanostructured surfaces even after surface area normalization.49
Figure 2.2: Proposed model of the effect of nanostructuring on DNA binding and hybridization. Nanostructured microelectrodes (NMEs) were used for electrochemical detection of DNA hybridization
with and without nanotexturing. Reprinted with permission from ref49. Copyright 2010 American
Chemical Society. 49
Other researchers have also demonstrated improved surface hybridization efficiencies due to nanostructured surfaces.1 Enhancement of electrochemical DNA hybridization
sensors has been shown using dendritic gold nanostructures,54 gold nanoflower-like
structures,55 gold-nanoparticle coated surfaces,56 other roughened gold surfaces,56,57 and
chemical nanostructuring and sub-nanometer structuring using mixed self-assembled monolayers (SAMS).58
As we can see, much of the work to date involving harnessing the geometric benefits of nanostructured surfaces on DNA hybridization has involved electrochemical sensors. There is good reason to believe, though, that it would exhibit enhancements in other detection methods as well, such as optical or piezoelectric-based transduction methods,
since increasing the number of species bound will increase the signal of any quantitative or semi-quantitative method.
Proteins
Like nucleic acids, the surface adsorption of proteins is influenced by surface nanostructuring. Unlike nucleic acids, proteins often exhibit a number of functional sites that can be bound to a surface, making control over their binding orientation both more difficult and more critical for subsequent recognition. For example, in immunosensing, involving recognition between an antibody and its corresponding antigen, it is required that the antibody be immobilized on a surface in an orientation that leaves the antigen binding site (Fab fragment) accessible.9 It is also critical to avoid protein denaturation or
conformational changes when binding proteins to a surface.59-61 There are a number of
methods that can be used to do this, as discussed already in a number of publications.7,9,59,62 In addition to immobilization in the proper orientation, it is important
to ensure that the density of bound proteins does not interfere with recognition ability. High protein densities on the surface can block the active sites of antibodies or other protein probes, preventing antigen binding.7,8
Surface nanostructuring can be a good way to ensure suitable binding densities and protein spacing. Work involving differently nanostructured arrays prepared by AFM nanografting of SAMs demonstrates the dependence of protein binding density and local environment on subsequent protein recognition; when arrays were designed according to the size of antibodies Fab sites, greater antibody recognition occurred10
(proposed model in Figure 2.3). A number of methods have also successfully been used to increase recognition efficiencies, including mixed SAMs giving chemically nanostructured surfaces,10,63,64 the use of dendrimers to create nanoscale spacing between
SAMs containing active groups,65 and nanostructured surfaces created by nanoparticle
Figure 2.3: Proposed model of the effect of nanostructuring on protein-antibody (biotin-IgG) interactions. Feature sizes similar to the size of the binding domains of IgG result in higher recognition (B, C). Feature sizes that are too small prevent recognition (A) and those that are too large result in random orientations
(D). Reprinted with permission from ref 10. Copyright 2008 American Chemical Society.
Another benefit of nanostructuring is an increased surface area available for probe binding. Rusling’s group claims this to be a contributing factor to the extremely low detection limits achieved in their electrochemical immunosensors featuring nanostructured surfaces using gold nanoparticles.67,68 The effects of these two
contributing mechanisms of enhancement—optimal protein density and increased surface area—can be difficult to separate, but the existing literature suggests that both play role in increasing analyte binding.
Enhancement of Optical Detection Methods
The plasmonic properties of gold surfaces and nanostructures have made them a major focus in current diagnostics research. Surface plasmons are electron cloud oscillations
that occur at the boundary between a metal and a dielectric. Waves of surface plasmons, known as surface plasmon polaritons, can be excited by photon or electron irradiation. Surface plasmon resonance (SPR) sensing makes use of changes related to these surface plasmon waves due to analyte binding for sensing applications including food quality and safety analysis, medical diagnostics, environmental monitoring, and drug discovery.13,69 In the case of nanosized and nanostructured materials, the surface
plasmon polariton is confined to a small area, smaller than the wavelength of the incident light, resulting in a phenomenon called localized surface plasmon resonance (LSPR). The wavelength for LSPR depends, among other factors, on the size of the nanostructure; when excited, it leads to enhanced light absorption and scattering. When these types of structures are used as substrates in techniques involving light absorption and scattering, such as Raman and infrared spectroscopy and fluorescence detection, they can electromagnetically enhance the detection signal, leading to phenomena such as surface-enhanced Raman scattering (SERS), surface-enhanced infrared absorption (SEIRA), and metal-enhanced fluorescence (MEF). Another related phenomenon involves electromagnetic hot-spots created at sharp tips and in small spaces between nanostructures, nanogaps, that can enhance optical processes, further increasing the enhancements seen in SERS, SEIRA, and MEF (Figure 2.4).33-36 While all related
phenomena have been used, both independently and simultaneously in interesting biosensing methods, the focus in this work regarding enhancement of optical methods will be on methods that make use of the latter phenomena—the creation of electromagnetic hot-spots and their use in diagnostic applications.
Several other reviews cover the general topic of optical enhancement by nanomaterials for biomedical applications in more detail.1,70,71
Figure 2.4: Simulations of electromagnetic enhancement at a) nanogaps72, b) sharp tips73, and c) combined
sharp tips and nanogaps (bowtie nanoantenna)74.
LSPR sensors
Localized surface plasmon resonance (LSPR) sensors offer some of the simplest set-ups in terms of optical methods making use of nanostructured surfaces. Sensing can require as little as the human eye, as is the case for colorimetric biosensing, or for more sensitive detection, a UV-visible spectrometer. LSPR sensing involves detecting changes in the refractive index, due to analyte binding, for example; a change in the refractive index causes changes in the frequencies needed for surface plasmon resonance. The greatest changes occur when binding occurs at electromagnetic hot-spots, such as nanogaps and sharp tips.37-39
A number of different formats have been used in LSPR sensing. Most LSPR sensors can be divided into either aggregation sensors or refractive index sensors.34 In aggregation
sensors, analyte presence induces metal nanoparticle aggregation, which results in a shift in the plasmonic peak of the particles.75-77 In refractive index sensors, analyte
presence induces a change in the refractive index of the dielectric medium at the surface of the metal, which also results in a plasmonic peak shift. A change in the refractive index at an electromagnetic hot-spot results in especially large shifts. A recent review
names preferential binding to these hot-spots as the next step in LSPR research,70 and to
date, some researchers have demonstrated hot-spot enhancement by preferentially binding biomolecules to hot-spot structures. Beeram and Zamborini demonstrated this by selectively binding anti-IgG to the edges of gold nanostructures on planar surfaces; the limit of detection when anti-IgG was selectively bound to electromagnetic hot-spots was at least 500 times lower than when not bound selectively.37 Feuz et al also
demonstrated this by selectively binding proteins to the hot-spot between two nanodisks and comparing the signal with binding to single gold disks. When normalized for surface area and thus signal per molecule, the signal is greater in nanogaps than on entire disks.38
SPR sensors
Surface plasmon resonance (SPR) sensors detect changes at a metal-dielectric interface by measuring changes in the conditions required to excite surface plasmons.69 Previous
work has shown that combining planar gold surfaces with plasmonic nanostructures can result in stronger signals and higher sensitivities.13 The enhancement is thought to be
due to coupling between surface plasmon polaritons (SPP) of the planar surface and localized surface plasmon resonance (LSPR) of the nanostructures.78,79 While the greatest
improvements have been seen using gold nanostructures as labels78,80-85 (as the presence
or absence of coupling is dependent on analyte binding), modest improvements have also been observed when gold nanoparticles are incorporated into the substrate.79,86-91
Nano- and micro-hole arrays offer another example of this coupling phenomenon.16
Holes in gold surfaces produce localized plasmons (similar to the nanogap enhancement observed between particles) and these are coupled with surface plasmons that propagate across the sample surface.
SERS
Surface-enhanced Raman spectroscopy, widely known as SERS, uses electromagnetic fields in metallic nanostructures to enhance the intensity of the signal in Raman
